Design and Synthesis of Multiple-Pyrene Labeled MBs
Previous DNA probes have incorporated pyrene derivates into various positions around a nucleotide in order to monitor pyrene-base and pyrene-pyrene interactions resulting from target binding.21–35
A method of assembling multiple quenchers using dendrimer linkers was applied here to assemble multiple pyrene molecules together at the 5′ end of a DNA sequence.36
With a single DABCYL (4-(4-(dimethylamino)phenylazo)benzoic acid) quencher molecule on the 3′ end, two to four pyrene molecules were linked to the 5′ end (MB-2P, MB-3P, and MB-4P) by varying the arrangement of carbon linkers (see Figure 8, supporting information). The pyrene labeled MB reported here (MB-2P) contains a dual-pyrene label on the 5′ end and a single DABCYL molecule on the 3′ end ().
Figure 1 (a) Structural representation of MB-2P with pyrene monomers labeled to dual 5′ linker chains and DABCYL molecule labeled to 3′ chain. (b) Schematic of MB-2P hybridization with complimentary target. (c) Emission spectra of 1 μM (more ...)
The dual-pyrene moiety positions the pyrene monomers in close proximity through two symmetric 18 atom linkers covalently attached at the 5′ terminal. schematically shows the hybridization of MB-2P with target DNA. In the beacon’s stem-open conformation, intense excimer fluorescence is generated as a result of the dimerization of the two adjacent pyrene monomers. Increasing the concentration of MB-2P in the hybridized state did not change the excimer/monomer emission ratio, confirming that dimer formation was due to intramolecular interactions. The long length of the alkane linkers is favorable, because it helps (i) minimize pyrene intercalation within the base stacking region of the DNA duplex and (ii) prevent monomer interactions with bases on the 5′ stem, thus avoiding fluorescence quenching effects. Unlike excimer-monomer switching mechanisms, the dual-pyrene functionality makes use of only the excimer emission which offers significant advantages over traditional fluorophores in spectroscopic signaling applications. Despite having more synthesis steps than traditional MBs, the strong hydrophobicity of the dual-pyrene label is remarkably effective in enhancing the purity of the MB, which in turn minimizes the residual fluorescence and target binding competition from non-labeled DNA sequences.
The DABCYL chromophore (λmax = 490 nm) was selected as a quencher, since it exhibits sufficient spectral overlap with the excimer emission of pyrene (480 nm) and provides an efficient non-radiative sink for the pyrene excimer. In addition, DABCYL has minor absorption in the pyrene monomer emission region, which helps further diminish the excimer emission, since the excited-state monomer is the precursor for the excimer state. DABCYL thus serves as an efficient excimer quencher in these multiple-pyrene MBs.
Steady-State Fluorescence Measurements to Detect Target DNA
To test the target signaling performance of MB-2P, steady-state fluorescence experiments were performed. shows the emission spectra of MB-2P at 350 nm excitation with varying concentrations of target DNA in buffer solution. The emission of MB-2P without target was indistinguishable from the background light scattering in the excimer emission range, indicating a high quenching efficiency by the DABCYL label. Two small peaks at 375 and 398 nm represented the fraction of unquenched monomer fluorescence in the dual-pyrene label. When titrated with target DNA, the excimer emission of MB-2P increased, reaching a maximum intensity with one equivalent of target. The spectra also show a slight, but steady increase in the emission peaks at 375 and 398 nm, which was due to the fluorescence of the pyrene monomer fraction being simultaneously restored when the MB hybridized to the target. As high as a 29-fold excimer signal enhancement was attained when equimolar target sequence was introduced into the MB solution. The sensitivity of this steady-state signaling mechanism is comparable with most FRET-based MBs with a limit of detection in the sub-nanomolar range. For example, a FAM labeled MB with the same sequence and quencher label exhibited a signal enhancement of approximately 15-fold under the same experimental conditions. The signal-to-background ratio of MB-2P was further enhanced by adding optimal amounts of divalent cations and DMF to the buffer. Hybridization results of MB-2P indicated that covalently bringing two pyrene molecules together allows efficient formation of an excimer complex. Furthermore, the excimer emission can be quenched in the same way as other organic fluorophores by common quencher molecules such as DABCYL. Thus, this pyrene assembly may be utilized in a variety of molecular probing platforms.
Time-Resolved Excimer Measurements
Differentiating the fluorescence signals between the probe and background species is often difficult with steady-state measurements. Alternatively, time resolved fluorescence monitoring can provide better signaling sensitivity in complex samples, but is dependent on the fluorescent lifetime and Stokes shift of the signaling agent. Lanthanide chelates (luminophores) are currently being explored as labeling agents in LRET-based probes due to their extraordinarily long fluorescence lifetimes and large Stokes shifts (up to 300 nm).37
Since most organic fluorophores possess lifetimes less than 10 ns, their use for such time-gated analysis is precluded. However, the pyrene excimer is unique among organic fluorophores, possessing a long fluorescence lifetime (40 ns) and large Stokes shift (130 nm). Retaining this lifetime and Stokes shift property in the multiple-pyrene label is critical for maximizing signaling sensitivity in complex biological samples.
Time-resolved fluorescence signaling of MB-2P was first investigated in buffer solution (). Without target, the fluorescence signal of the quenched dual-pyrene label was retained longer than the buffer signal at both 398 and 480 nm. The background emission increase of MB-2P was noticeably more pronounced in the monomer channel due to both a weaker monomer quenching efficiency (less absorption overlap from DABCYL molecule at 398 nm) and substantially longer emission lifetime of the pyrene monomer (100 ns) compared to the light scattering from the buffer (< 7 ns). The lifetime of MB-2P at 480 nm was 33 ns (70% fluorescence contribution) with most of the signal originating from the unquenched excimer fraction in the dual-pyrene label. In the presence of excess cDNA, the fluorescence decay of MB-2P changed very little in the monomer channel (), whereas the excimer emission intensity increased significantly over the full time scale (). The unquenched excimer emission of the dual-pyrene label was easily differentiated from the buffer and background emissions over a sustained time period after excitation. A maximum signal-to-background enhancement of 25 was achieved in the 90–100 ns time interval. The lifetime of target-hybridized MB-2P increased to 39 ns (87.5%), indicative of the unquenched excited-state of the pyrene excimer in solution. This value is approximately one order magnitude longer than the fluorescence lifetimes of most biological species, thus making time-resolved fluorescence signaling feasible in complex biological samples.
Figure 2 Fluorescence decay traces after 337 nm excitation of 1 μM MB-2P (blue) and MB-2P with target DNA (red) in buffer solution. Fluorescence emissions were monitored at a) the pyrene monomer wavelength (398 nm) and at b) the pyrene excimer wavelength (more ...)
Nucleic Acid Detection in Cell Media
To evaluate its potential effectiveness in bioassays, MB-2P was tested in Dulbecco’s cell growth media. shows the steady-state emission spectra of MB-2P in the presence and absence of target in both buffer solution and cell growth media.
Steady-state emission spectra of 500 nM MB-2P comparing background fluorescence in cell media and buffer. Excimer signal enhancement in cell media is poor compared to buffer.
In the cell growth media, the high autofluorescence dominated the luminescence spectra from 400 to 500 nm. To discriminate the dual-pyrene excimer emission from the high background fluorescence, time-gated photon counting was employed. compares the emission intensities of MB-2P in the presence of target at different times after excitation. At 0 ns, the spectrum is dominated by the autofluorescence of the medium and resembles the steady-state luminescence spectrum shown in . At 40 ns, the spectrum closely resembled the steady-state spectrum of MB-2P in buffer with a dominant pyrene excimer emission peak at approximately 480 nm. At 100 ns after the excitation pulse, the luminescence spectrum is dominated by the pyrene monomer fluorescence, because of the longer fluorescence lifetime of the pyrene monomer compared to the excimer.
Figure 4 (a) Time-resolved emission spectra of 500 nM MB-2P with 5 μM cDNA in cell growth media. The fluorescence emission was recorded at 0 ns (black), 40 ns (red), and 270 ns (blue) time gates, (b) Emission decay at 480 nm of 500 nM MB-2P (blue) and (more ...)
The sensitivity of the time-resolved excimer signaling approach was investigated for quantifying DNA in cell growth media. It followed from the time-resolved emission spectra that once the autofluorescence had decayed away (less than 40 ns after excitation), a local emission maximum for MB-2P was observed in the pyrene excimer emission channel. The fluorescence decays of MB-2P were thus appropriately monitored at this channel (480 nm) with varying concentrations of target cDNA in the cell media (). The total photon counts increased proportionally with cDNA concentration and a clear signal separation was obtained for the fully hybridized MB-2P. A maximum signal separation was observed in a time window of 60 – 110 ns. In this time window, much of the excimer emission still occurred, while most of the background autofluorescence had decayed. These results show time-resolved analysis of the excimer fluorescence to be a sensitive and robust method for detecting low nanomolar DNA concentrations in complex biological media without the need for sample pretreatment, extractions, or target amplification.
Tunable Intensity through Multiple-Pyrene Labeling
Another feature of the multiple-pyrene label is that the excimer intensity can be scaled,34
to a large extent, by the local pyrene density at the terminal of the MB. Unlike pyrene, other fluorophores will self-quench if intermolecular distances are too short. It has been shown that double-labeling organic fluorophores to the terminals of free oligonucleotides significantly decreases the fluorescence intensity relative to single-fluorophore labeled oligonucleotides.15
While most commercial organic fluorophores have Stokes shifts ranging from 10 and 40 nm, the pyrene excimer exhibits an exceedingly long Stokes shift (approx. 130 nm) with no overlap between the monomer absorption (350 nm) and excimer emission (480 nm) bands. This peak separation allows the pyrene excimer to avoid the effects of self-quenching even at high concentrations and labeling densities. More importantly, pyrene excimer formation requires the complexation of an excited-state molecule with a ground state molecule. Increasing the density of pyrene molecules assembled at the MB terminal enhances the probability of forming excited-state dimers. In polypyrenylalkanes, it has been shown that intramolecular excimer formation is favored both by the thermodynamic stabilization of the dimer and the long excited-state lifetime of the pyrene monomer during which excimer formation proceeds.20
In the dendrimeric pyrene assemblies reported here, the long length and symmetric branching arrangement of the alkane linkers provide more flexibility for the pyrene molecules to interact equivalently and thus not be limited to nearest-neighbor interactions, such as with rigid, linear assemblies. This may further enhance excimer formation since any one of the pyrene monomers, when excited, can dimerize with any of the ground-state monomers with equal probability. The expanded excimer intensity range of these multiple-pyrene MBs may offer improved signaling sensitivity in fluorescence-based detection methods.
In addition to MB-2P, we synthesized MBs with 1,3, and 4 pyrene monomers conjugated at the 5′ end to investigate the effect of pyrene assemblies on the excimer signaling performance in MBs.
The emission spectra of the pyrene labeled beacons in the presence of excess cDNA showed a substantial increase in excimer intensity with an increased number of pyrene monomers in the label (). The monomer emission intensity at 376 and 398 nm also increased, but to a much lesser extent than the excimer emission for the MBs bearing 2, 3, and 4 pyrenes. The increased excimer/monomer emission ratio for each additional pyrene () indicates excimer formation was more favorable with higher densities of pyrene labeled at the terminal. The fluorescence quantum yields of the different MBs were determined (). The quantum yield increased with an increasing number of pyrenes in the label, reaching a value of 0.20 for MB-4P. Such a high quantum yield, together with a remarkable extinction coefficient (56,000×4=224,000 cm−1M−1), make the 4-pyrene label an exceptionally bright UV excitable fluorophore. The fluorescence decays were also monitored to investigate if the increased excimer signals from the MBs were sustained after pulse excitation. shows the fluorescence decays at 480 nm for the multiple-pyrene MBs in the presence of excess cDNA. Each additional pyrene monomer resulted in slower excimer decay, with an increase in fluorescence lifetime of 8 ns for MB-4P relative to MB-2P (). This increased excimer lifetime has the potential to provide higher background discrimination, and thus higher signaling sensitivity, in both steady-state and time-resolved bioassays.
a) Steady-state emission spectra of MB-1P (black), MB-2P (blue), MB-3P (red), and MB-4P (green) with excess cDNA in buffer, b) Fluorescence decay of MB-2P (black), MB-3P (red), and MB-4P (green) at 480 nm. [MB]=200 nM, [cDNA]=2μM
Photophysical and performance properties of MBs with different numbers of pyrene molecules in the label.
Hybridization experiments indicate that although overall excimer intensity increased with the number of pyrene molecules, the background signal from the free MB also increased, as the steady-state signal-to-background ratio of MB-4P was nearly three fold lower than that of MB-2P (). It was apparent that the excimer quenching efficiency of the single DABCYL molecule decreased as the number of pyrene monomers increased. This was likely due in part to a larger overall separation between the pyrene and quenching moieties, which reduced the energy transfer efficiency. Another potential reason may be that the emission intensity exceeded the quenching capacity of the single DABCYL molecule. Two possibilities for improving the quenching efficiency in the stem-closed MBs include (i) matching the lengths of the linkers connecting the quencher and pyrene molecules and (ii) replacing the single quencher with a superquencher assembly36
to increase the number of available energy acceptors. These modifications will be evaluated in future work.
These results suggest a way to build a bright fluorophore by assembling multiple pyrene molecules together in a way that both preserves probe functionality and enhances excimer formation. While previous pyrene assemblies have been made use of linear polypeptide35
backbones, these dendrimeric linkages bring multiple pyrene monomers together in close proximity to further enhance the probability of excimer formation. Of course, solubility of the product may be a problem for some biological applications because of the hydrophobicity of the pyrene assembly. Therefore, water soluble pyrene derivatives, such as Cascade Blue, may be better suited for these pursuits. Our initial testing indicates that this fluorophore exhibits similar excimer forming properties as pyrene with the excimer emission peak around 510nm (Figure 9, Supporting Information). Also, to alleviate the quenching of pyrene by oxygen common to biological environments, alkynylpyrene derivatives38
have been shown to retain high fluorescence intensity in aerated environments and thus have potential to be employed in similar excimer signaling probes as presented here.
Stem Stability Analysis
Considering the long length of the dendrimeric linkers and hydrophobicity of the pyrene molecules, the ability of the MB stem to open freely in the presence of cDNA was likely to be impacted by a combination of stabilizing and destabilizing interactions between the multiple-pyrene moiety and either the quencher moiety or bases within the stem helix.
We evaluated the rate of stem opening by comparing the hybridization kinetics of MB-1P and MB-4P in buffer solution (). The excimer fluorescence intensity was monitored vs. time for each MB (monomer fluorescence for MB-1P and excimer fluorescence for MB-4P) in the presence of complimentary target. As shown in , under the same conditions, the half-time to open MB-1P was approximately 76 seconds while MB-4P required 71 seconds. This result suggests the multiple-pyrene labels have little effect on the hybridization kinetics of the MB to its target DNA. A thermal denaturation analysis () was also conducted to compare the relative stem stabilities in each MB. The excimer fluorescence intensity was monitored with increasing temperature as the equilibrium shifted from the stem-closed to stem-open conformation (monomer emission used for MB-1P). The melting point for MB-1P was 60.1 °C and approximately 1.5 °C lower than any of the multiple-pyrene MBs. There was no clear difference in stability, however, between the MBs containing multiple pyrenes conjugated to the terminal. This indicates that the multiple-pyrene assemblies may be self-aggregating and thus causing the stem stability to be independent of additional pyrene monomers in the label (beyond 2). In addition, the multiple pyrene label may exhibit more affinity toward the quencher moiety than a single pyrene linker, which may account for the small difference in melting temperatures between the single and multiple pyrene MBs.
Figure 6 a) Hybridization kinetics of MB-1P and MB-4P. Three hybridization trials were conducted for each beacon (a,b,c) [MB]=100nM, [cDNA]=1μM. b) Thermal denaturation profiles of multiple-pyrene labeled beacons. Monomer intensity (376 nm) was monitored (more ...)
For bioanalysis applications, fluorophores with long Stokes shifts are ideal for resolving the fluorescence signal against the shorter-wavelength autoluminescence.
To extend the emission wavelength beyond the pyrene excimer emission, multiple-pyrene-fluorophore assemblies were explored as alternative FRET-based signaling agents that employ the pyrene excimer as a fluorescence donor. Tetramethylrhodamine (TMR) was chosen as a fluorescence acceptor due to its absorption in the 500–550 nm region coinciding with the excimer emission range. Two compounds were synthesized each containing 1 TMR molecule and 2 or 3 pyrene monomers respectively attached to linkers spaced 17 atoms from a central carbon atom. A compound containing 1 pyrene monomer and 1 TMR molecule was also synthesized as a control. The close distance of each dye within the complex is to ensure a high probability of both pyrene excimer formation and FRET between the excimer and acceptor fluorophore. shows the steady-state emission spectrum of the pyrene-TMR complexes excited at 344 nm. Major emission peaks at 590 nm were observed for both the 2P-1TMR and 3P-1TMR complexes. The pyrene monomer and excimer emission peaks were also visible, although the excimer emission intensity was low relative to the monomer and TMR intensities. This suggests that FRET may be occurring between the pyrene excimer state and TMR thus resulting in a combined Stokes shift of more than 240 nm. For the control complex, 1P-1TMR, there was no excimer emission and only minor TMR emission was observed due to direct excitation. This indicates that FRET was not feasible when only a single pyrene monomer was in range of the acceptor fluorophore. Multiple-pyrene conjugates incorporating other red-emitting fluorophores, such as Texas Red, may also have a similar FRET capability and provide an even larger Stokes shift between excitation and acceptor emission. These results suggest the potential application of multiple-pyrene assemblies as fluorescent donors in FRET-based molecular probes. The increased separation between the pyrene excitation and fluorescence channels provided by suitable acceptor fluorophores will make it easier to discriminate between true and false-positive signals in complex biological media.
Figure 7 (a) Chemical structure of 3-pyrene-TMR conjugate, (b) Emission spectra of pyrene-TMR conjugates excited at 345 nm in buffer and normalized to the pyrene monomer emission to approximate equal concentrations. The spectra of 2 and 3-pyrene conjugates reveal (more ...)